Extended-spectrum ß-lactamases (ESBLs) are a worldwide public health problem: (7)they have rapidly emerged worldwide in Enterobacteriaceaethey are considered to be one of the most important antibiotic resistancemechanisms

The majority of ESBLs belong to TEM-, SHV- and CTX-M types:(http://www.lahey.org/studies/)

More than 180 TEM-type and 130 SHV-type β-lactamases have beenidentified worldwideThe main mutations conferring the ESBL phenotype are found at the followingpositions:

TEM: 104, 164, 238 and 240SHV: 238 and 240

All the CTX-M enzymes are ESBLs: more than 90 CTX-M variants, divided into5 five groups (CTX-M-1, 2, 9 and 8/25), have been identified (2).

Resistance to carbapenems due to the production of KPC enzymes inEnterobacteriaceae is a growing issue as well (3). To date, 11 KPC-variants have beendescribed.

Optimal detection of ESBLs/KPC is now highly important: (4)ESBLs detection is primarily based on phenotypic testing and standardmolecular tests tend to be used to characterize isolates with these genes.PCR-sequencing is the most widely method used.Detection of KPC enzymes is difficult using phenotypic methods

The huge diversity of these enzymes makes their detection truly crucial to routinelymonitor their prevalence and worldwide distribution. Fast and reliable moleculartechniques which could be used in clinical microbiology laboratories are essential.

Introduction

1. Performance Standards for Antimicrobial Susceptibility Testing; Nineteenth Informational Supplement. CLSIdocument M100-S19, 2009. Clinical and Laboratory Standards Institute (CLSI), Wayne, PA 19087-1898USA.

2. Bonnet, R. 2004. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. AntimicrobAgents Chemother 48:1-14.

3. Cuzon, G., T. Naas, and P. Nordmann. [KPC carbapenemases: what issue in clinical microbiology?]. Pathol Biol (Paris)58:39-45.

4. Jones, C. H., A. Ruzin, M. Tuckman, M. A. Visalli, P. J. Petersen, and P. A. Bradford. 2009. Pyrosequencing using thesingle-nucleotide polymorphism protocol for rapid determination of TEM- and SHV-type extended-spectrum beta-lactamases in clinical isolates and identification of the novel beta-lactamase genes blaSHV-48, blaSHV-105, andblaTEM-155. Antimicrob Agents Chemother 53:977-86.

5. Mulvey, M. R., E. Bryce, D. Boyd, M. Ofner-Agostini, S. Christianson, A. E. Simor, and S. Paton. 2004. Ambler classA extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. in Canadian hospitals.Antimicrob Agents Chemother 48:1204-14.

6. Nuesch-Inderbinen, M. T., H. Hachler, and F. H. Kayser. 1996. Detection of genes coding for extended-spectrumSHV beta-lactamases in clinical isolates by a molecular genetic method, and comparison with the E test. Eur J ClinMicrobiol Infect Dis 15:398-402.

7. Pitout, J. D., and K. B. Laupland. 2008. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: anemerging public-health concern. Lancet Infect Dis 8:159-66.

8. Speldooren, V., B. Heym, R. Labia, and M. H. Nicolas-Chanoine. 1998. Discriminatory detection of inhibitor-resistant beta-lactamases in Escherichia coli by single-strand conformation polymorphism-PCR. AntimicrobAgents Chemother 42:879-84.

9. Woodford, N., E. J. Fagan, and M. J. Ellington. 2006. Multiplex PCR for rapid detection of genes encoding CTX-Mextended-spectrum (beta)-lactamases. J Antimicrob Chemother 57:154-5.

References

#C-126

IHMA, Inc.2122 Palmer Dr.

Schaumburg, IL 60173Tel: 847.303.5003www.ihmainc.com

clascols@ihmainc.com

Results

A Novel DNA Microarray Method for Rapid Detection and Identification of Extended-spectrum β-lactamases (ESBLs) and KPC in EnterobacteriaceaeC. Lascols1, R. Badal1, D. Hoban1, C. Cruz1, K. Szewczyk1, M. Hackel1, S. Hawser2, S. Bouchillon1 and J. Johnson1

1International Health Management Associates, Schaumburg, IL, USA2IHMA Europe Sàrl, Epalinges, Switzerland

Check-Points combined to PCR-sequencing:

Figure 4: Procedure scheme for this study

Confirmation of the Check-Points results byPCR-sequencing

Figure 5: Check-Points results

Bacterial strains:

The Study for Monitoring Antimicrobial Resistance Trends (SMART) is a globallongitudinal antimicrobial surveillance study that has been monitoring susceptibility ofGram-negative aerobic pathogens from intra-abdominal infections (IAI) since 2002

In this study, 1,185 recent isolates (2008-2009) from the SMART programwere tested.1,126 clinical isolates phenotypically identified as ESBL-positive59 clinical isolates phenotypically identified as ESBL-negative (negative controls)ESBL testing was done according to CLSI guidelines (1), looking for a >3-doublingdilution decrease in MIC of ceftazidime or cefotaxime in the presence of clavulanic acid. 7 control strains, representing the ESBL targeted (SHV, TEM, CTX-M1, 2, 9, 8/25)by the Check-Points method and PCR, were used in this study.

Materials & Methods

Background: Extended spectrum β-lactamases (ESBLs) of the TEM, SHV, or CTX-Mtype and KPC-producers have rapidly emerged worldwide. Fast and reliable detectiontechniques are essential in order to assist choice of therapy and to limit resistancespread. We used a novel in vitro molecular diagnostic test "Check-Points (CP) Check-KPC ESBL " for the direct identification of ESBL (TEM, SHV and CTX-M) and KPC-producers on a large panel of Enterobacteriaceae clinical isolates. Methods: Multicenterworldwide collection of Escherichia coli, Proteus mirabilis, Klebsiella spp and Enterobacterspp ESBLs (n=1,126) and non-ESBL (n=59) isolates collected from the SMARTprogram (2008-2009) were tested. Whole DNA extracts were submitted to aligation reaction with specific probes. Ligated probes were subsequently amplifiedusing a universal set of primers. Amplicons were specifically hybridized to the DNAmicro-array and then processed with CP software. Results were confirmed by PCR-sequencing. Results: The CP assay allowed fast and unambiguous detection of all ESBL,non-ESBL and also KPC genes. Further CP assay identifies the point mutation in TEMand SHV genes that confer the ESBL phenotype and allows therefore differentiationbetween non-ESBL TEM and SHV and their ESBL derivatives. This is particularly usefulfor E. coli and K. pneumoniae. Of the 1,126 ESBL-positive isolates tested, 1,080 weredetected by the CP array and all ESBL-negative isolates were negative by CP. In thisstudy, the CP array identified 16 TEM, 198 SHV and 943 CTX-M ESBL genesrespectively and 27 KPC genes as well. All the ESBL SHV, TEM and CTX-M genesdetected by CP were confirmed by PCR. Sequencing data were 96% correlated with theCP results for all the SHV, TEM and CTX-M genes. Conclusion: This novel assay is a high-throughput powerful tool for rapid detection and categorization of ESBLs and KPC-producers. This method offers differentiation of ESBLs from non-ESBLs and definitiveresults within the same working day. The panel of genotypes detected has beenadvantageously extended to other genes (AmpC, carbapenemases).

Revised Abstract

Check-Points array :

Is a powerful tool for the detection ofESBL-phenotype and identification of:

- TEM types- SHV types-CTX-M group 1, 2, 9 and 8/25

identifies ESBL from non-ESBL TEMand SHV

is more adapted for batch testing inclinical lab equipped for molecular biology

can be combined with PCR-sequencing,for fast, accurate and completecharacterization of ESBLs

is truly a promising tool for thedetection of ESBL (SHV, TEM, CTX-M)but also carbapenemases and AmpCenzymes.

Conclusions

We sincerely thank the laboratory personnel: M. Person, C. Cruz, C. Gaylord, M. McCarthy, G.Ronkova and K. Szewczyk for performing microbiological testing.

Acknowledgments

The 46 negative results by CP were due to CTX-M1 “weaksignals” not read by the CP software but which could be read bythe experimenter. Recently, the CP software has been updated andthe CTX-M1 probe has been improved.All KPC detected by Check-Points have been confirmed by PCR-sequencing (27 isolates)

Figure 6: Sequencing results

Discrepancies between Check-Points results(codes) and sequences:

Table 2: SHV genes

Table 3: TEM genes

Advantages of Check-ESBL KPC combined withPCR-sequencing

Figure 7: Example for an isolate carrying only the CTX-M1 enzyme:

The Check-Points array results allowed avoidingsequencing of the non-ESBL enzymes.

Figure 8: Time to response for complete characterizationof 72 isolates

3 working days for complete characterization of72 isolates

Correlation : 96%

DNA sample preparation:

Whole DNAs were extracted with theQIAGEN QiaAmp DNA mini kitusing the QIAcube instrument(12 samples an hour)

DNA micro-array method:

Figure 1: Principle of the CP method:

A-Ligation Detection Reaction

Probe contains:Probe Arm 1:A specific sequence at the 5’ with universal primers (blue box) linked to a unique ZIPcode (orange box)Probe Arm 2:A specific sequence at the 3’ with universal primers (yellow box)

Probes are hybridized to target DNA and only in case of a perfect match, the probesare joined by a ligase. Critical mismatches in the target sequence will cause ligation tofail, leaving the probes apart.

B-Polymerase Chain reaction (PCR)

Successful ligation products are amplified by PCR using a single pair of universalprimers annealing to complementary sequences included in the probes (blue and yellowboxes)

C-Hybridization and Detection

Unique ZIP codes (orange box) assigned to each probe will be specifically captured bycomplementary oligonucleotides spotted on the microarray and will be detected usinga biotin label incorporated in one of the PCR primers.

The final results are obtained using aspecific reader (ATR03) and software:

The system can be multiplexed with many differentprobes, each bearing a different ZIP code.

PCR-sequencing:Primers used for detection and sequencing of the blaCTX-M, blaSHV, blaTEM and

blaKPC were described previously (5, 6, 8, 9)Both strands of the PCR products were sequenced using an Applied Biosystemssequencer (ABI 3730)Sequences were analyzed using the SeqScape software

Material & Methods(cont.)

Interpretation of Check-ESBL KPC results

Table 1a: Example of correlation of Check-ESBL scores and the SHV genes

Table 1b: Example of correlation of Check-ESBL scores and the TEM genes

Figure 2: ESBL-KPC array layout

Figure 3: Check-KPC ESBL software

Material & Methods(cont.)